The mammalian immune system has evolved to defend the organism against pathogenic microbes, layering the specificity of adaptive responses on the ancestral pathways of innate immunity. This complexity exists to provide discrimination between self and non-self and to insure that immune responses are tightly regulated, thus avoiding autotoxicity and uncontrolled inflammation. Multiple checkpoints have been identified that function to insure an orderly progression through an immune response and thereby prevent the generation of self destructive processes. A common theme that has emerged from the study of these checkpoints is the requirement for the establishment of discrete thresholds that define narrow windows of response. One mechanism to achieve these thresholds is for the co-expression of receptors with common ligand binding properties but divergent signaling capacities, coupling activating receptors with an inhibitory counterpart thereby setting thresholds for immune cell activation (Ravetch, Fc receptors. In Fundamental Immunology, W. E. Paul, ed. (Philadelphia, Lippincott-Raven), pp. 685-700 (2003)).
Although cellular receptors for immunoglobulins were first identified nearly 40 years ago, their central role in the immune response was only discovered in the last decade. They are key players in both the afferent and efferent phase of an immune response, setting thresholds for B cell activation and antibody production, regulating the maturation of dendritic cells and coupling the exquisite specificity of the antibody response to effector pathways, such as phagocytosis, antibody dependent cellular cytotoxicity and the recruitment and activation of inflammatory cells. Their central role in linking the humoral immune system to innate effector cells has made them attractive immunotherapeutic targets for either enhancing or restricting the activity of antibodies in vivo.
The interaction of antibodies and antibody-antigen complexes with cells of the immune system effects a variety of responses, including antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC), phagocytosis, inflammatory mediator release, clearance of antigen, and antibody half-life (reviewed in Daron, Annu Rev Immunol, 15, 203-234 (1997); Ward and Ghetie, Therapeutic Immunol, 2, 77-94 (1995); Ravetch and Kinet, Annu Rev Immunol, 9, 457-492 (1991)), each of which is incorporated herein by reference).
Antibody constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins (isotypes): IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses, e.g., IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called α, δ, ∈, γ, and μ, respectively. Of the various human immunoglobulin classes, human IgG1 and IgG3 mediate ADCC more effectively than IgG2 and IgG4.
Papain digestion of antibodies produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual “Fc” fragment, whose name reflects its ability to crystallize readily. The Fc region is central to the effector functions of antibodies. The crystal structure of the human IgG Fc region has been determined (Deisenhofer, Biochemistry, 20, 2361-2370 (1981), which is incorporated herein by reference). In human IgG molecules, the Fc region is generated by papain cleavage N-terminal to Cys, 226.
Several antibody functions are mediated by Fc receptors (FcRs), which bind the Fc region of an antibody. FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors for IgG antibodies are referred to as FcγR, for IgE as Fc∈FR, for IgA as FcαR and so on. Surface receptors for immunoglobulin G are present in two distinct classes—those that activate cells upon their crosslinking (“activation FcRs”) and those that inhibit activation upon co-engagement (“inhibitory FcRs”).
In all mammalian species studied to date, four different classes of Fc-receptors have been defined: FcγRI (CD64), FcγRII (CD32), FcγRIII (CDI6) and FcγRIV. Whereas FcγRI displays high affinity for the antibody constant region and restricted isotype specificity, FcγRII and FcγRIII have low affinity for the Fc region of IgG but a broader isotype binding pattern (Ravetch and Kinet, 1991; Hulett and Hogarth, Adv Immunol 57, 1-127 (1994)). FcγRIV is a recently identified receptor, conserved in all mammalian species with intermediate affinity and restricted subclass specificity (Mechetina et al., Immunogenetics 54, 463-468 (2002); Davis et al., Immunol Rev 190, 123-136 (2002); Nimmerjahn et al., (2005)).
Functionally there are two different classes of Fc-receptors: the activation and the inhibitory receptors, which transmit their signals via immunoreceptor tyrosine based activation (ITAM) or inhibitory motifs (ITIM), respectively (Ravetch, in Fundamental Immunology W. E. Paul, Ed. (Lippincott-Raven, Philadelphia, (2003); Ravetch and Lanier, Science 290, 84-89 (2000). The paired expression of activating and inhibitory molecules on the same cell is the key for the generation of a balanced immune response. Additionally, it has only recently been appreciated that the IgG Fc-receptors show significant differences in their affinity for individual antibody isotypes rendering certain isotypes more strictly regulated than others (Nimmerjahn et al., 2005).
The mouse expresses three activation FcγRs, FcRI, FcRIII and FcRIV, oligomeric surface receptors with a ligand binding α subunit and an ITAM containing γ subunit. The inhibitory receptor is FcγRIIB, a single chain receptor with an ITIM sequence found in the cytoplasmic tail of the ligand binding α chain. FcRIIB and FcRIII bind monomeric IgG with an affinity constant of 1×106; hence, under physiological conditions they do not bind monomeric IgG, but interact with multimeric IgG immune complexes with low affinity and high avidity. FcRIII and FcRIV are physiologically important activation FcRs for mediating inflammatory disease triggered by cytotoxic antibodies or pathogenic immune complexes. FcRIII is expressed on dendritic cells, NK cells, macrophages, monocytes, mast cells and neutrophils in the mouse, while FcRIV is found on dendritic cells, macrophages, monocytes and neutrophils. They are not found on B cells, T cells, red blood cells or platelets. FcRIIB is found on most hematopoeitic cells, including dendritic cells, B cells, macrophages, monocytes mast cells and neutrophils. It is not found on T cells or NK cells. FcRII and III have greater than 90% sequence identity in their extracellular, ligand binding domain, while FcRIV is most homologous to human FcRIIIA
The situation in the human is analogous. There are three low-affinity activation FcRs for IgG-FcγRIIA, FcγRIIC and FcγRIIIA. FcγRIIA and FcγRIIC are a single-chain low affinity receptors for IgG, with an ITAM sequence located in their cytoplasmic tail. They are expressed on dendritic cells, macrophages, mast cells, monocytes and neutrophils. They are 90% homologous in their extracellular domains to the human inhibitory FcRIIB molecule, which has an ITIM sequence in its cytoplasmic domain, expressed on dendritic cells, B cells, macrophages, mast cells, neutrophils, monocytes but not NK cells or T cells. FcRIIIA is an oligomeric activation receptor consisting of a ligand binding subunit and an ITAM containing γ or ζ subunit. It is expressed on NK cells, macrophages and mast cells. It is not expressed on neutrophils, B cells or T cells. In addition, a receptor with greater than 95% sequence identity in its extracellular domain called FcRIIIB is found on human neutrophils as a GPI-anchored protein. It is capable of binding immune complexes but not activating cells in the absence of association with an ITAM containing receptor like FcRIIA. FcRII and FcRIII are about 70% identical in their ligand binding extracellular domains.
Thus, in the human, IgG antibodies interact with four distinct low-affinity receptors—three of which are capable of activating cellular responses, FcRIIA, FcRIIC and FcRIIIA, one of which is inhibitory, FcRIIB and one of which will bind IgG complexes but not trigger cellular responses, FcRIIIB. Macrophages expresses FcRIIA, FcRIIB and FcRIIIA, neutrophils express FcRIIA, FcRIIB and FcRIIIB, while NK cells express only FcRIIIA. The biological activity of an IgG antibody will thus depend on the specific interactions with activation, inhibition and inert low-affinity FcRs, differentially expressed on distinct cell types.
Diversification of IgG subclasses is most strikingly observed in mammals where detailed characterization of four subclasses has been described (Litman et al., Annu Rev Immunol 17, 109-47 (1999)). In both rodents and primates these subclasses display differential abilities to mediate effector responses, such as antibody dependent cytotoxicity, phagocytosis and release of inflammatory mediators (Burton and Woof, Adv Immunol 51, 1-84 (1992); Ravetch, (2003) pp. 685-700; Ravetch and Bolland, Annu Rev Immunol 19, 275-90 (2001)). Skewing of the expression of IgG subclasses is regulated by both the antigen and cytokine milieu, such that IL-4 preferentially induces switching to IgG1 and IgE, while TGF-β induces switching to IgG2b and IgA (Finkelman et al., Annu Rev Immunol 8, 303-33. (1990); Stavnezer, J Immunol 155, 1647-51 (1995); Snapper and Mond, Immunol Today 14, 15-7 (1993)). Thymic dependent antigens primarily result in IgG1, 2a and 2b responses; in contrast, thymic independent antigens typically lead to IgG3 accumulation (Mond et al., Curr Opin Immunol 7, 349-54 (1995)). Further distinctions among the subclasses occurs in response to T cell derived responses with TH1 cytokines resulting in IgG2a, 2b and 3 switching, while TH2 cytokines lead to IgG1 and IgE dominated responses (Mosmann, and Coftman, Annu Rev Immunol 7, 145-73 (1989)). Among the IgG subclasses, IgG2a and 2b are generally considered to be the most potent at activating effector responses and have been found to dominate in both anti-viral and autoimmune conditions (Coutelier et al., J Exp Med 165, 64-9 (1987); Markine-Goriaynoff and Coutelier, J Virol 76, 432-5. (2002); Fossati-Jimack et al., J Exp Med 191, 1293-302 (2000); Uchida et al., (2004)).
Immune complexes consisting of IgG antibodies bind to activating Fc receptors (FcR) and inhibitory FcRs that are expressed by innate immune effector cells such as basophils, mast cells, neutrophils, monocytes and macrophages, in which they trigger the indicated effector responses. Binding of immune complexes to FcRs on dendritic cells results in phagocytosis and presentation of antigenic peptides on MHC class I and class II molecules. Antigen-specific CD8+ cytotoxic T cells, CD4+ helper T cells or regulatory T cells (TReg cells) that recognize these peptide-MHC complexes become activated and mediate various effector functions such as killing of virus-infected cells, modulation of immune responses or providing T-cell help for antigen-specific B cells. B cells only express inhibitory low-affinity FcR for IgG (FcγIIB), which regulates activation signals transduced by the B-cell receptor (BCR). On plasma cells, which produce high levels of antigen-specific antibodies, BCR expression is very low or absent, resulting in exclusive triggering of inhibitory signaling pathways which can result in apoptosis on those cells.
The family of Fc receptors (FcRs) for IgG (FcγRs) provides a prime example of how simultaneous triggering of activating and inhibitory signaling pathways sets thresholds for cell activation and thus generates a well-balanced immune response. (Ravetch & Lanier, Science 290:84-89 (2000)). Indeed, in a variety of human autoimmune diseases, such as arthritis and systemic lupus erythematosus (SLE), aberrant expression or the presence of allelic variants of FcγRs with altered functionality have been observed that contribute to the pathogenesis of these diseases. In particular, expression of the inhibitory FcγRIIB receptor on B cells has been linked to susceptibility to autoimmune diseases such as lupus by altering B cell homeostasis and thereby contributing to the loss of tolerance to self antigens.
The inhibitory FcγRIIB is the most broadly expressed FcγR, and is present on virtually all leukocytes with the exception of NK cells and T cells. Because of the broad expression pattern, it is not surprising that genetic deletion of this negative regulator results in complex phenotypic changes affecting innate and adaptive immune responses.
Antibody binding to cellular FcγRs efficiently induces pro-inflammatory responses that lead to the removal of virus-infected or malignant cells, but it can also lead to the destruction of healthy tissues during autoimmune responses. Therefore, antibody specificity, as well as class switching to antibody isotypes that efficiently trigger pro-inflammatory reactions through their interaction with cellular FcγRs, have to be tightly controlled. Groundbreaking work over the last few years has established that several central and peripheral checkpoints exist throughout B-cell development to prevent the generation of autoreactive antibodies (Goodnow et al, Nature 435, 590-597 (2005)). On a molecular level, gene-deletion studies in mice have been instrumental in identifying several proteins (including the inhibitory FcγRIIB) that are involved in regulating B-cell activity.
One common theme that emerged from these studies is the importance of the ITIMs found in the cytoplasmic domains of these proteins (Amigorena et al, Science 256, 1808-1812 (1992); and Muta et al, Nature 369, 340 (1994)). Simultaneous triggering of ITIM-containing proteins with the BCR results in the recruitment of phosphatases such as SHIP (SH2-domain-containing inositol polyphosphate 5′ phosphatase) and SHP1 (SH2-domain-containing protein tyrosine phosphatase 1) that interfere with activating signalling pathways by hydrolysing phosphoinositide intermediates (Bolland. & Ravetch, Adv. Immunol. 72, 149-177 (1999). Nitschke. & Tsubata, Trends Immunol. 25, 543-550 (2004), Ono et al, Nature 383, 263-266 (1996), Ono et al, Cell 90, 293-301 (1997)). This prevents the recruitment of pleckstrin homology (PH)-domain-containing kinases, such as BTK or PLCy, to the cell membrane, thereby diminishing downstream events such as the increase in intracellular calcium levels. Thus, deletion of these regulatory proteins results in a lower threshold for B-cell activation and stronger activating signals after BCR crosslinking (Nitschke. & Tsubata, (2004)).
The importance of the inhibitory FcγRIIB in modulating B-cell activity and humoral tolerance is supported by studies of mice and humans. Decreased or absent expression of FcγRIIB resulted in the development or exacerbation of autoimmune diseases and several mechanisms responsible for this reduced expression were identified. Regardless of the model system studied, FcγRIIB has emerged as a late checkpoint during peripheral B-cell development that acts at the level of class switched B cells or antibody producing plasmablasts or plasma cells. Given the incomplete purging of autoreactive B cells from the immature repertoire in the bone marrow and the de novo generation of these cells during the process of affinity maturation, late peripheral checkpoints are of utmost importance.
Recent data suggests that the inhibitory FcγRIIB is important for regulating plasma-cell survival itself. The first evidence that the isolated triggering of FcγRIIB can induce apoptosis in B cells was reported 10 years ago. Co-engagement of the BCR and FcγRIIB in SHIP deficient B cells induced apoptosis (Ono et al, (1996), Ono et al, (1997)). Similarly, the homo-oligomerization of FcγRIIB resulted in increased levels of B-cell death, and it was shown later that a signaling pathway dependent on BTK, JNK1 and cABL, but independent of SHIP and ITIM, was responsible for this phenotype (Pearse et al, Immunity 10, 753-760 (1999) and Tzeng et al, J. Biol. Chem. 22, 22 (2005)). It was suggested that this scenario might arise during the germinal-centre reaction, in which B cells are in close contact with immune complexes presented on the surface of FDCs. Whereas B cells that generate a higher-affinity BCR will receive signals from both the BCR and FcγRIIB, B cells that lose affinity for the cognate antigen will only receive signals through FcγRIIB and will therefore be deleted.
Another situation in which a B cell expresses virtually no BCR and high levels of FcγRIIB is the terminally differentiated plasma cell. Plasma cells reside predominantly in niches in the bone marrow, where they have to receive survival signals from stromal cells (Radbruch et al, Nature Rev. Immunol. 6, 741-750 (2006)). If deprived of these anti-apoptotic signals, plasma cells rapidly die owing to pro-apoptotic signals triggered by a constant endoplasmic-reticulum-stress response induced by the continuous production of antibodies. One current conundrum is how the limited number of niches available in the bone marrow can accommodate the vast number of antigen-specific plasma cells that are necessary to protect the body from all types of pathogens (Radbruch et al, (2006)). How newly generated plasma cells gain access to these niches has remained a matter of debate, and models such as competitive dislocation have been proposed to explain this problem (plasma blasts and mobilization of resident plasma cells in a secondary immune response. (Odendahl et al, Blood 105, 1614-1621 (2005)). The pro-apoptotic signals triggered by isolated FcγRIIB crosslinking by immune complexes on plasma cells might be another elegant solution to this problem (Ravetch & Nussenzweig, Nature Immunol. 8, 337-339 (2007); and Xiang et al, Nature Immunol. 8, 419-429 (2007)). Immune complexes generated de novo during an immune response could bind to plasma cells in the bone marrow and induce apoptosis on a fraction of cells, thus making space for newly generated plasma cells. Indeed, secondary immunizations with a new antigen result in reduced numbers of bone marrow plasma cells that are specific for the primary antigen (Xiang et al, (2007)).
Interestingly, plasma cells from autoimmune-prone mouse strains show absent or strongly reduced expression of FcγRIIB, and are resistant to induction of apoptosis. By contrast, restoration or overexpression of the inhibitory receptor could correct this defect (Xiang et al, (2007)). Therefore, the failure to control plasma-cell persistence resulting from impaired FcγRIIB expression levels might account for their large number in autoimmune-prone mouse strains and ultimately for the development of chronic autoimmune disease. Correction of FcγRIIB expression levels might be a promising approach to interfere with autoimmune processes and to restore tolerance.
Therefore, in autoimmune conditions, such as lupus or rheumatoid arthritis, hyperactivation of B cells is observed with inappropriate production of autoantibodies. These B cells have escaped from the normal regulation imposed by the inhibitory FcRIIB receptor. Co-engagement of activation cell surface B cell molecules with FcRIIB would address this problem and provide a means to reduce B cell activation. Methods and means to accomplish this are presented below.
DCs are the most potent antigen-presenting cells and can efficiently prime cellular immune responses. Besides this well-established function it is has become clear that during the steady state, these cells are actively involved in the maintenance of peripheral T-cell tolerance (Ono et al, Nature 383, 263-266 (1996)). Thus, targeting antigens to DCs in vivo without the addition of co-stimulatory signals, such as those that trigger CD40 or Toll-like receptors, leads to the deletion or functional inactivation of antigen-specific CD4+ and CD8+ T cells (Dudziak et al, Science 315, 107-111 (2007); Hawiger et al, J. Exp. Med. 194, 769-779 (2001); Hawiger et al, Immunity 20, 695-705 (2004); Kretschmer et al, Nature Immunol. 6, 1219-1227 (2005); and Steinman et al, Ann. NY Acad. Sci. 987, 15-25 (2003)). This suggests that potentially self-reactive T cells that escaped deletion by central tolerance mechanisms in the thymus, will be rendered inactive upon recognition of self antigens on DCs in the periphery. In addition, there is evidence that antigen presentation to CD4+ T cells by DCs under tolerogenic conditions can induce regulatory T cells de novo (Kretschmer et al, (2005)). Therefore, the maturation state of DCs has to be tightly controlled to prevent both the initiation of self-destructive responses and the generation of regulatory T cells during a protective antimicrobial immune response. A great number of activating and inhibitory cell surface proteins involved in the regulation of DC activation have been identified (Schuurhuis et al, Int. Arch. Allergy Immunol. 140, 53-72 (2006)). Among them, the family of activating and inhibitory FcγRs has been shown to be of central importance for setting a threshold for DC activation and in the modulation of the adaptive cellular immune responses. This, however, is not the only function of FcγRs on DCs, as they are also important for endocytosis and/or phagocytosis of immune complexes and presentation of antigen-derived peptides on MHC molecules (Woelbing et al, J. Exp. Med. 203, 177-188 (2006)). Therefore, FcγRs control three functions that are of central importance to any immune response initiated by DCs: antigen uptake, antigen presentation and cell activation.
Most FcγRs can only interact with antibodies in the form of immune complexes resulting in high-avidity binding. During an active immune response, a large number of immune complexes are generated owing to the priming of antigen-specific B cells. Several studies have shown that immune complexes are potent activators of DCs and are able to prime stronger immune responses than antigen alone (Regnault et al, J. Exp. Med. 189, 371-380 (1999); Dhodapkar et al, J. Exp. Med. 195, 125-133 (2002); 88. Groh et al, Proc. Natl. Acad. Sci. USA 102, 6461-6466 (2005); 89. Rafiq et al, J. Clin. Invest. 110, 71-79 (2002); and 90. Schuurhuis et al, J. Immunol. 176, 4573-4580 (2006). Importantly, FcγR-dependent immune-complex internalization not only resulted in MHC-class-II-restricted antigen presentation but also in cross-presentation on MHC class I molecules, thereby priming both CD4 and CD8 T-cell responses (Regnault et al, (1999)). The magnitude of this response is controlled by the inhibitory FcγRIIB, as DCs derived from Fcgriib-knockout mice generate stronger and longer-lasting immune responses in vitro and in vivo (Bergtold et al, Immunity 23, 503-514 (2005); and Kalergis & Ravetch J. Exp. Med. 195, 1653-1659 (2002)). More importantly, FcγRIIB deficient DCs or DCs incubated with a monoclonal antibody that blocks immune complex binding to FcγRIIB showed a spontaneous maturation (Boruchov et al, J. Clin. Invest. 115, 2914-2923 (2005); and Dhodapkar et al, Proc. Natl. Acad. Sci. USA 102, 2910-2915 (2005)). This implies that the inhibitory FcγR not only regulates the magnitude of cell activation but also actively prevents spontaneous DC maturation under non-inflammatory steady-state conditions. Indeed, low levels of immune complexes can be identified in the serum of healthy individuals, emphasizing the importance of regulatory mechanisms that prevent unwanted DC activation (Dhodapkar et al, (2005)).
In situations in which a maximal immune response is desirable, such as immunotherapy of malignancies or microbial infections, blocking FcγRIIB activity might be a novel way to obtain greater therapeutic efficacy. Along these lines, it has been demonstrated that the genetic deletion of the gene encoding FcγRIIB results in the priming of more antigen-specific T cells (Kalergis & Ravetch (2002)). Moreover, current approaches for targeting antigens to DCs in vivo by genetic fusion of the antigen to an antibody Fc fragment rely on an antibody mutant that does not bind to FcγRs to prevent FcγR-mediated modulation of cell activity. With the availability of antibody variants with enhanced binding to either activating or inhibitory FcγRs, however, it might become possible to integrate an additional activating or inhibitory second signal into the antibody-antigen fusion protein (Lazar et al, Proc. Natl. Acad. Sci. USA 103, 4005-4010 (2006); and Shields et al, J. Biol. Chem. 276, 6591-6604 (2001)). Depending on the application, this would permit the generation of either tolerogenic or immunogenic responses without adding secondary reagents. As will be discussed later, mouse models in which the mouse FcγRs have been replaced with their human counterparts (referred to as FcγR humanized mice) will be essential to test these optimized antibody variants in vivo.
In addition to its expression on B cells, the inhibitory FcγRIIB is expressed on innate immune effector cells, such as mast cells, granulocytes and macrophages. As these cells have the capacity to trigger strong pro-inflammatory responses, their activation needs to be tightly controlled. In the case of antibody-mediated responses, such as phagocytosis, ADCC, allergic reactions and release of pro-inflammatory mediatiors, this is the function of the inhibitory FcγRIIB. This crucial role is exemplified by enhanced macrophage responses in Fcgriib-knockout mice in models of collagen-induced arthritis and immune-complex-mediated alveolitis. (Clynes et al., J. Exp. Med. 189:179-185 (1999); Yuasa et al, J. Exp. Med. 189:187-194 (1999)).
Recent studies have demonstrated that the inhibitory receptor contributes a varying level of negative regulation depending on the specific IgG subclass that is bound to the receptor. (Nimmerjahn & Ravetch, Science 310, 1510-1512 (2005)). This is consistent with the observation that different IgG subclasses have different activities in vivo. For example, in a variety of mouse model systems, IgG2a or IgGb antibody subclasses are more active than IgG1 or IgG3. (Nimmerjahn et al., Immunity 23, 41-51 (2005)). Whereas IgG1 shows the strongest level of FcγRIIB-mediated negative regulation, the activity of IgG2a and IgG2b was increased less dramatically by the absence of this receptor. (Nimmerjahn & Ravetch, (2005)). This can be explained by the differences in the affinity of these isototypes for the different activating and inhibitory FcγRs. This ratio of affinities of a given IgG subclass for the activating versus the inhibitory receptor has been termed the A/I-ratio and it has emerged as a good predictive value for the activity of a specific IgG subclass in vivo (Nimmerjahn et al., Immunity 23, 41-51 (2005); Nimmerjahn & Ravetch, (2005)). These studies indicate that the effector cells responsible for mediating the activity of the different IgG subclasses express both activating and the inhibitory FcγRs. As NK cells lack FcγRIIB expression this argues against a role for NK cells as the responsible effector cell in mice in vivo. Indeed, myeloid cells that abundantly express FcγRIIB have been suggested to be the responsible effector-cell type in models of ADCC and SLE (Uchida et al., J Exp Med 199, 1659-69 (2004); Begtold, J. Immunol. 177, 7287-7295 (2006)).
Accordingly, there is an immediate need for improved reagents, methods and systems for designing therapeutic antibodies and vaccines for treatment of autoimmune disease.